Chemical reprogramming of human glial cells into neurons with small molecule cocktail

ABSTRACT

Provided are compositions, articles and methods that relate to promoting neurogenesis or neuroregeneration in mammalian nervous system. Embodiments relate to use of groups of compounds that contain Crizotinib (Cri), Flurbiprofen, Lithium Chloride (Li), Vitamin C (VC), Ceritinib (Cer) or Pirfenidone (PFD). In certain implementations glial cells are converted into functional neurons.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.62/263,353, filed Dec. 4, 2015, the disclosure of which is incorporatedherein by reference.

FIELD

The present disclosure relates generally to prophylaxis and therapy ofconditions related but not limited to neural injury, neurodegeneration,aging, microcephaly, severe seizure resulting in neuronal loss, and morespecifically to compositions and methods comprising combinations ofchemicals for converting internal glial cells into functional neuronsfor neural repair, neuroregeneration, and neuroprotection.

BACKGROUND

Brain disorders such as stroke and Alzheimer's disease do not haveeffective therapies that can reverse their progression largely becauseof a lack of methods for regenerating sufficient numbers of new neuronsfor brain repair. The cell transplantation therapy using external cellsto transplant into the brain or spinal cord (Buhnemann et al., 2006;Emborg et al., 2013; Nagai et al., 2010; Nakamura and Okano, 2013; Okiet al., 2012; Sahni and Kessler, 2010) have faced significant hurdlesincluding immunorejection, tumorigenesis and differentiation uncertainty(Lee et al., 2013; Liu et al., 2013b; Lukovic et al., 2014). Recentstudies have used viruses to express transcription factors in cells toconvert them into neurons both in vitro and in vivo (Heinrich et al.,2010; Grande et al., 2013; Torper et al., 2013; Guo et al., 2014; Niu etal., 2013; Su et al., 2014). Some studies have used chemicals to convertfibroblast cells into neurons, which still need to be transplanted intothe brain or spinal cord, facing all the difficulty of celltransplantation including immunorejection, tumorigenesis anddifferentiation uncertainty (Hu et al., 2015; Li et al., 2015). Usingglial cells for neural conversion offers great advantages because theyare resident cells throughout the nervous system, and are different fromstem cells that are rather limited inside the nervous system. Anotheradvantage is that glial cells can divide and regenerate themselves.Therefore, if some glial cells are converted into neurons, the remainingglial cells have the potential to divide and generate new glial cells.The present disclosure is pertinent to a need for methods for promotingconversion of glial cells into neurons.

SUMMARY

In the Figures and description of this disclosure Ceritinib isabbreviated as Cer, Pirfenidone is abbreviated as PFD, Crizotinib isabbreviated as Cri, Flurbiprofen is abbreviated as Flu, Lithium Chlorideis abbreviated as Li, and Vitamin C is abbreviated as VC. In certainreferences to groups of compounds CC refers to Crizotinib and Certinib.F refers to Flurbiprofen. L refers to Lithium Chloride. V refers toVitamin C. P refers to Pirfenidone. Each of these compounds is wellknown in the art, is commercially available, and each compound isindividually approved by the U.S. Food and Drug Administration (FDA) foradministration to humans for certain indications, which currently do notinclude any of the presently disclosed uses. The approaches embodied inthis disclosure thus differ greatly from previous methods, at least inpart because they involve reprogramming of glial cells using chemicallysynthesized compounds that are already FDA approved for use in humans.As such it does not include the risks associated with introducingexogenous genes, viral vectors, or engineered cells into patients, nordoes it require manipulating stem cells or other multipotent cells inculture to differentiate them into neurons or otherwise prepare thecells for administration to a subject. Instead, the instant disclosureencompasses reprogramming glial cells already present in the individualsuch that they are converted into neurons using combinations of smallmolecules that are more fully described below. The compositions andmethods are expected to provide a convenient and safe approach to treata variety of conditions that involve neuronal deficiency, for example,neuronal loss after injury, neurodegeneration, aging, microcephaly, orsevere seizure resulting in neuronal loss. It will be recognized bythose skilled in the art that neural injury can result from a number ofcauses that are known in the art, and which typically involveastrogliosis after injury or disease processes in the central nervoussystem including brain and spinal cord, and peripheral nervous system.Reactive astrocytes are the main cellular component of glial scars,followed by NG2 glia and microglia. Thus, in embodiments, the presentdisclosure comprises converting astrocytes into neurons by chemicallyinduced reprogramming of the astrocytes. But similar chemicalreprogramming methods may also be used to convert NG2 glia or microgliaor other cell types surrounding brain blood vessels into neurons.

In certain embodiments the disclosure comprises administering acombination of drugs selected from the group consisting of Ceritinib(Cer), Pirfenidone (PFD), Crizotinib (Cri), Flurbiprofen, LithiumChloride (Li), Vitamin C (VC), and combinations thereof. Ceritinib is ananaplastic lymphoma kinase (ALK) inhibitor. Pirfenidone is a TGF-betasynthesis inhibitor. Crizotinib inhibits ALK and c-MET. Flurbiprofen isa γ-secretase inhibitor. LiCl is a Glycogen synthase kinase 3 (GSK3)inhibitor. Thus, there are a total of six agents that have been testedin furtherance of the present disclosure. In particular embodiments, thedisclosure includes administering a combination of Cri/Li/Flu, plus atleast one additional agent selected from PFD, Cer, VC. Groups meetingthis criteria and that are demonstrated to function in the presentdisclosure comprise the following combinations; i) Cer/Cri/Li/Flu/VC,ii) PFD/Cri/Li/Flu/VC, iii) Cer/Cri/Li/Flu, and iv) PFD/Cri/Li/Flu. In anon-limiting demonstration the group comprises or consists ofCer/Cri/Flu/PFD/VC/Li. Any of these groups can be considered necessaryand sufficient for the conversion, but the disclosure includes for eachof these combinations also including at least one of the other membersof the group of six agents that are not included in each group.

This disclosure includes pharmaceutical compositions comprising orconsisting of any group of compounds described herein. Also provided arearticles of manufacture, e.g., kits, comprising compounds and printedmaterial. The printed material provides an indication that the compoundsare for use in treating an individual in need of neurogenesis. Theindividual may be in need of neurogenesis for a wide variety ofconditions and/or because of neuronal injury.

BRIEF DESCRIPTION OF THE FIGURES

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-FIG. 11. Results showing the indicated groups of FDA-approveddrugs reprogram cultured human glial cells into neurons in vitro. FIG.1A, Combination of Crizotinib, Certinib, Flurbiprofen, Lithium Chloride,and Vitamin C converted human glial cells into neurons. FIG. 1B,Combination of Crizotinib, Flurbiprofen, Lithium Chloride, Pirfenidone,and Vitamin C converted human glial cells into neurons. FIG. 1C,combination of Crizotinib, Flurbiprofen, Lithium Chloride, andPirfenidone converted human glial cells into neurons. FIG. 1D,combination of Crizotinib, Certinib, Flurbiprofen, and Lithium Chloride,converted human glial cells into neurons. FIG. 1E, vehicle control 0.2%DMSO, the solvent for Cer, PFD, Cri and Flu, showed very few NeuN+ cellsafter 14 days. FIG. 1F, Quantitative analyses showing significantincrease of NeuN+ cell numbers after drug-treatment. FIG. 1G.Characterization of human astrocytes with the immunostaining ofastrocyte-specific marker glutamate transporter 1 (Glt1) (Green, FIG.1G), and human nuclei marker HuNu (Blue, FIG. 1H), and the overlay image(FIG. 1I).

FIG. 2A-FIG. 2B. Results showing that neurons converted by the indicatedcompounds can survive at least 2 months in culture. Human neuronsconverted from astrocytes after CCFLV (FIG. 2A) or CFLPV (FIG. 2B)treatment survived at least 2 months in culture, and showed matureneuronal markers MAP2 (light blue) and NeuN (Red). DAPI (dark blue)labels cell nucleus.

FIG. 3A-FIG. 3G. Data showing intracranial injection of the indicatedFDA-approved drugs increases hippocampal adult neurogenesis in the adultmouse brains. FIG. 3A shows that an intracranial injection of 2 μlsmall-molecule cocktail including Crizotinib 50 μM, Flurbiprofen 0.2 mM,Pirfenidone 2 μM, Vitamin C 10 mg/ml and LiCl 0.4 M into the hippocampusof 3-month-old WT mice, promoted adult neurogenesis revealed byimmunostaining of newborn neuron marker doublecortin (DCX, green) andcell proliferation marker Ki67 (red). FIG. 3B. Intracranial injection ofsmall molecule cocktail into the hippocampus of 1-year-old adult WTmice, significantly promoted adult neurogenesis in dentate gyrus (DG),supported by the increased number of DCX-positive new neurons andKi67-positive proliferating cells. FIG. 3C. Intracranial injection ofsmall molecule cocktail into the hippocampus of 5-month-old transgenicmouse model of Alzheimer's disease, significantly increased the numberof DCX-positive new neurons in DG. FIG. 3D. In GFAP::GFP mice,astrocytes are labeled by GFP. Lineage-tracing assay indicate the DCX+new neurons were induced from GFP-labeled astrocytes by small-moleculecocktail. FIG. 3E, FIG. 3F, and FIG. 3G present quantitative analysisshowing the number of DCX+ new neurons was increased by intracranialinjection of small-molecule cocktail into the hippocampus of 3-month-oldWT mice (FIG. 3E), 1-year-old adult WT mice (FIG. 3F), and 5-month-oldtransgenic mice model of Alzheimer's disease (FIG. 3G). Student t test,*P<0.05, **P<0.01, n=3 mice per group.

FIG. 4A-FIG. 4C. Data demonstrating that intraperitoneal injection ofthe indicated FDA-approved drugs can also increase adult neurogenesis inthe mouse brains. (FIG. 4A, FIG. 4B) Typical images showing adultneurogenesis in the hippocampal dentate gyrus after intraperitonealinjection of vehicle control (FIG. 4A, 20% Captisol), or small-moleculecocktail (FIG. 4B) including Crizotinib 50 μM, Flurbiprofen 0.2 mM,Pirfenidone 2 μM, Vitamin C 10 mg/ml and LiCl 0.4 M (3-month-old WT micewith the dose of 0.1 ml/10 g weight, injected daily for 1 month). Sevendays after chemical treatment, mice were sacrificed and examined withimmunostaining of newborn neuron marker DCX. (FIG. 4C) Quantitativeanalysis revealed an increased number of DCX+ neurons treated by theFDA-approved drug cocktail. Student's t test, *P<0.05, n=2 pairs.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise herein, all technical and scientific terms usedin this disclosure have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure pertains.

Every numerical range given throughout this specification includes itsupper and lower values, as well as every narrower numerical range thatfalls within it, as if such narrower numerical ranges were all expresslywritten herein.

The present disclosure comprises compositions and methods that aredesigned to convert human glial cells into functional neurons. Inembodiments the disclosure comprises but is not necessarily limited togeneration of new neurons from endogenous glial cells, and can includegenerating new neurons from glia-like cells created due to injury or adisease condition in the central or peripheral nervous system usingindicated compounds, which is expected to be useful for a variety oftherapies, non-limiting embodiments of which include brain and spinalcord repair. The method generally comprises administering to anindividual in need thereof an effective amount of a combination ofcompounds selected from the group comprising or consisting of Ceritinib(Cer), Pirfenidone (PFD), Crizotinib (Cri), Flurbiprofen (Flu), LithiumChloride (Li), Vitamin C (VC), and combinations thereof. There areaccordingly a total of six agents that have been tested as will bedescribed more fully below. The disclosure includes administering anyone or any combination of these agents. In embodiments, the combinationincludes two, three, four, five or all six of the compounds, and mayinclude other compounds. In embodiments the disclosure relates to use ofa combination of Cri/Li/Flu, plus at least one additional agent selectedfrom PFD, Cer, VC.

As described in the summary above, in particular embodiments, thedisclosure includes administering a group of drugs that comprise orconsist of the following combinations; i) Cer/Cri/Li/Flu/VC, ii)PFD/Cri/Li/Flu/VC, iii) Cer/Cri/Li/Flu, and iv) PFD/Cri/Li/Flu. Thedisclosure includes for each of these combinations the option ofincluding at least one of the other members of the group of six agentsthat are not included in each of the four enumerated groups.Administration of the compounds to individuals is expected to result inat least some glial cells in the individual being converted intoneurons. In embodiments, alternative compounds are used, where suchcompounds have the same or similar effect as the compounds listed above,and wherein the administration of the combination results in conversionof glial cells into neurons. In embodiments, conversion into neuronstakes place over a period of approximately 7 to 14 days.

In embodiments, the disclosure is expected to be broadly applicable fortherapy of any human subject in need of neuronal generation. The needfor neuronal generation arises as a consequence of any of a variety ofconditions, disorders or injuries that affect neuronal function, and/orreduce the number of functional neurons in the individual. Thus, thedisclosure is pertinent to prophylaxis and/or therapy of conditionswhich include but are not necessarily limited to ischemic brain damage,such as that caused by stroke, hypoxia or other brain trauma, or glialscarring, or neurodegeneration, or aging, or microcephaly, or severeseizure that causes neuronal loss. In embodiments the disclosure ispertinent to treating neurodegenerative disorders, including but notlimited to Alzheimer's disease or other conditions which present withdementia, or Chronic Traumatic Encephalopathy (CTE) such as in athleteswith a history of acute or repetitive brain trauma (i.e., concussions),or Parkinson's Disease, or Huntington's disease, or multiple sclerosis,or glioma, or spinal cord injury, or spinal muscular atrophy, orAmyotrophic lateral sclerosis (ALS). In a non-limiting embodiment ademonstration of in vivo neurogenesis promotion is demonstrated in ananimal model of Alzheimer's disease.

The present disclosure does not include introduction of modified cellsor viral constructs into a subject, and moreover there is no requirementfor administering stem cells to an individual. For example, it isexpected that aspects of the current approach will not require in vitrodifferentiation of cells, including stem cells, and importantly, priorart processes are distinct from our reprogramming of glial cells toneuronal cells, because stem cells can differentiate naturally intoneurons but glial cells cannot become neurons unless subjected to areprogramming process such as that demonstrated in this disclosure.Further, those skilled in the art will recognize that injecting culturedstem cells or their differentiated neurons into human subjects, andespecially the brain poses significant risk to the host. Likewise, thepresent glia-to-neuron conversion technology is also distinct from thoseconverting fibroblast cells into neurons in vitro, because glial cellsare internal cells inside the nervous system, whereas fibroblast cellsare not. Therefore, the fibroblast-converted neurons have to betransplanted into the human subject, whereas the present glia-to-neuronconversion is in situ, without any need of transplantation. In addition,as described above, it has been demonstrated that astroglial cells canbe converted into neurons in vivo, but such approaches involveintroduction of viral vectors, or other exogenous genes into thesubjects which pose particular risks to the subject.

The present disclosure provides in various embodiments the use ofcompletely cell and virus free pharmaceutical formulations that comprisechemical compounds that act in concert with one another to coax glialcells or glia-like cells to convert to neurons. The present disclosureprovides an in vivo demonstration that these compounds can pass throughthe blood-brain-barrier through systemic administration and act insidethe brain.

In embodiments, the disclosure comprises administering to a subject inneed thereof an effective amount of one or more compositions comprisingas an active ingredient a combination of compounds that are selectedfrom Cer, PFD, Cri, Flu, Li, VC, and combinations thereof. In one aspectthe disclosure includes administering a combination of Cri/Li/Flu, plusat least one additional agent selected from PFD, Cer, VC. Particular andnon-limiting groups of compounds that are encompassed by the disclosureinclude: i) Cer/Cri/Li/Flu/VC, ii) PFD/Cri/Li/Flu/VC, iii)Cer/Cri/Li/Flu, and iv) PFD/Cri/Li/Flu. In non-limiting embodimentsgroups of these compounds are shown to promote neurogenesis in mousebrain using both intracranial and intraperitoneal administrations. Inembodiments the group of compounds comprises or consists ofCri/Flu/PFD/VC/Li.

Each of these compounds is known in the art and is commerciallyavailable. The disclosure includes compositions and methods thatcomprise groups of any three, four, five, or six of these compounds, andmay include additional compounds as described herein or as wouldotherwise be apparent to one skilled in the art, given the benefit ofthe present disclosure. The disclosure includes pharmaceuticallyacceptable salts of these compounds, analogs of the compounds and salts,and compounds which exert the same or similar functions as thecompounds, provided that administration of a combination of them to anindividual results in conversion of glial cells to neurons. In generalreference to any particular compound described herein for use ofconversion of glial cells to neurons includes pharmaceuticallyacceptable salts of the compounds.

In an embodiment, the disclosure includes administering to an individuala combination of compounds (concurrently or sequentially), wherein thecombination comprises or consists of Cri/Li/Flu, plus at least one, oronly one, additional agent selected from PFD, Cer, VC.

It will be apparent from the description, examples and figures of thisdisclosure that we have discovered that in combination the smallmolecules as described herein are capable of directly reprogramminghuman astrocytes into functional neurons. Because these chemicals areapproved by FDA for the treatment of humans or can be obtained over thecounter in drug stores, they are relatively safe and are expected toresult in a convenient approach to chemical delivery for therapy of awide variety of neural injuries and neurodegenerative conditions,including but not necessarily limited to stroke and Alzheimer's disease.

In general, methods of the disclosure comprise administering aneffective amount of the compounds described herein to a subject suchthat the number of neurons in the individual is increased. The compoundscan be administered in amounts that are the same or similar to those forwhich FDA approval is already in place. Dosages for each of the FDAapproved drugs can be found, for example, inwww.accessdata.fda.gov/scripts/cder/drugsatfda/. In embodiments, glialcells, such as astrocytes in the individual are reprogrammed so thatthey are converted into neurons. In embodiments, the newly generatedneurons comprise glutamatergic neurons. In embodiments, the disclosureis expected to facilitate development of new cortical forebrain neurons,or midbrain neurons, or hindbrain neurons, or spinal cord neurons, orperipheral neurons, or combinations thereof by using methods describedherein adapted as necessary by those skilled in the art in a manner thatwill be apparent given the benefit of the present disclosure. Inembodiments, reprogrammed neurons are characterized by expression ofneuronal markers that include but are not necessarily limited to Dcx andNeuN. In embodiments, cells in the brain, such as glial cells, areconverted to neurons. In embodiments, the neurons are functionalneurons. Functional neurons can exhibit properties which can comprisebut are not necessarily limited to firing repetitive action potentials,developing a plurality of dendritic branches, and release ofneurotransmitters, including but not necessarily limited to Glutamate(glutamic acid), dopamine, acetylcholine, serotonin, Norepinephrine(noradrenaline), and γ-Amino butyric acid (GAB A).

Data presented in this disclosure demonstrate at least the following:Groups of compounds that comprise Cri/Li/Flu and at least one of Cer, VCor PFD are effective in generating human neurons from human glial cellsin vitro. In particular, each of the groups i) Cer/Cri/Li/Flu/VC, ii)PFD/Cri/Li/Flu/VC, iii) Cer/Cri/Li/Flu and iv) PFD/Cri/Li/Flu not onlyconverted human astrocytes into neurons (FIGS. 1A-1F), the convertedneurons were able to survive in culture for at least two months (FIGS.2A and 2B). FIG. 1G provides a characterization of human astrocytes withthe immunostaining of the astrocyte-specific marker glutamatetransporter 1 (Glt1) (Green, FIG. 1G), and human nuclei marker HuNu(Blue, FIG. 1H), and the overlay image (FIG. 1I).

Furthermore, the disclosure provides non-limiting demonstrations of suchan approach in vivo. In particular, both intracranial (FIG. 3) andintraperitoneal administration (FIG. 4) results in an increase inneurogenesis in mammalian brain. The disclosure demonstrates achievingthis not only in adult mouse brain, but also in the brain of a mousethat has been engineered to model Alzheimer's disease (e.g., FIG. 3C).Thus, the disclosure provides working examples of promoting in vivoneurogenesis in mammalian brain. Moreover, and without intending to bebound by any particular theory, it is believed that the disclosureproves that there is sufficient crossing of the blood-brain barrier(e.g., the i.p. administration of FIG. 4) such that the presentdisclosure is suitable for a variety of routes of administration. Incertain and non-limiting embodiments, in vivo neurogenesis using anadministration that is not limited to intracranial administration ispromoted using the group of compounds comprising or consisting ofCri/Flu/PFD/VC/Li. But based on the data of this disclosure taken as awhole, it is expected that groups of compounds that comprise at leastthree of Cri/Li/Flu/Cer/VC/PFD will also promote neurogenesis withoutbeing limited to intracranial administration. In embodiments, Cri/Li/Fluand at least one of Cer, VC or PFD will also promote neurogenesis inmammalian brain without being limited to intracranial administration.Also without being constrained by any particular theory, it isconsidered that mouse data presented herein, particularly when viewed incombination with the results obtained with human cells, provides a basisfor predicting that in vivo administration of groups of compounds ofthis disclosure to humans will be effective in promoting neurogenesis inhuman brain.

Compositions comprising the compounds of this disclosure can be providedin pharmaceutical formulations. The form of pharmaceutical preparationis not particularly limited, but generally comprises these activeingredients and at least one inactive ingredient. In certain embodimentssuitable pharmaceutical compositions can be prepared by mixing any oneor combination of the compounds with a pharmaceutically-acceptablecarrier, diluent or excipient, and suitable such components are wellknown in the art. Some examples of such carriers, diluents andexcipients can be found in: Remington: The Science and Practice ofPharmacy (2005) 21st Edition, Philadelphia, Pa. Lippincott Williams &Wilkins. In embodiments, the pharmaceutical formulations are suitablefor delivering the active ingredients across the blood-brain barrier,and/or to the spinal cord or other components of the central nervoussystem. Such compositions can comprise, for example, lipid formulationsor other nano-particle based delivery systems.

In one embodiment the pharmaceutical formulation is suitable for oraladministration, and thus can be provided in an aerosolized, liquid orsolid dosage form. Solid dosage forms include but are not necessarilylimited to tablets, capsules, caplets, and strips, for swallowing ororal dissolution, and may be provided for rapid or extended release, orto release distinct compounds in a desirable series over a period oftime. Separate pharmaceutical compositions comprising one or anycombination of the compounds can also be used. Thus the pharmaceuticalformulations can comprise Cri/Li/Flu, plus at least one additional agentselected from PFD, Cer, VC. It should be understood that any of thecompounds described herein can be excluded from the compositions andmethods of the invention.

With respect to the administration of the pharmaceutical formulations,the route of administration can be any suitable route. In embodiments,the composition comprising the compound(s) is delivered orally. In othernon-limiting embodiments, the composition is administered intravenously,parenterally, subcutaneously, intraperitoneally, transdermally, byintranasal instillation, by implantation, or intraarterially. Inembodiments, an implantable medical device can be used, such as a pump,including but not limited to an osmotic pump. In embodiments thecompositions comprising the compounds is delivered via an intracranialroute.

Appropriate dosing of the compound(s) can be determined in conjunctionwith the knowledge of the skilled artisan, given the benefit of thepresent disclosure. In embodiments, the weight and age of theindividual, personal history of neuronal damage or disease and risk forexperiencing same neuronal damage, or the presence of glial scarring orreactive gliosis, may be taken into account when determining aneffective amount of the active ingredient and dosing regimen. Inembodiments the compounds are administered in an amount of about 0.01nmol to about 500 nmol a day, inclusive, and including all integers andranges there between, depending on which delivering method being used.In embodiments, the compounds are provided in a single, multiple, orcontrolled release dose regimen.

In certain embodiments Ceritinib is used in a concentration of about 50nM, which may be adjusted when administered to human patients to achievea concentration in human blood of approximately 5-500 nM. Forpirfenidone, a suitable concentration may be approximately 5 nM. Forcrizotinib, suitable concentration may be approximately 125 nM. ForFlurbiprofen, suitable concentration may be approximately 500 nM. ForLiCl a suitable concentration may be approximately 2 mM. For Vitamin C,suitable concentration may be approximately 50 ug/mL.

In certain embodiments the disclosure includes nutraceuticalcompositions, which are designed to impart to an individual a beneficialeffect that is related to improved neuronal health and/or function. Incertain embodiments, the compositions of the invention can be used toimprove the general well-being of an individual, or the cognitivecapability of an individual, such as for improved memory or maintenanceof memory. In embodiments the compositions are useful for improving anyor all of short term memory, long term memory, or motor skills,including but not necessarily limited to gross and fine motor skills.Thus, use of nutritional supplements comprising the small moleculesdescribed herein are encompassed by this disclosure.

In one embodiment, the disclosure includes an article of manufacture. Incertain aspects, the article of manufacture includes a closed or sealedpackage that contains one or a combination of the compounds describedherein, such as in separate tablets, capsules or the like. The packagecan comprise one or more containers, such as closed or sealed vials,bottles, blister (bubble) packs, or any other suitable packaging for thesale, or distribution, or use of pharmaceutical agents. Thus, thepackage can contain pharmaceutical compositions which Cri/Li/Flu, plusat least one additional agent selected from PFD, Cer, VC, and/or othercompounds that are described herein and as otherwise be recognized assuitable additions by those skilled in the art. Any one or all of thesecompounds can be included, and each can be provided separately or incombination with one or more of the others in the same or distinctdosage formulations so that they can be delivered concurrently, orsequentially.

In addition to the pharmaceutical compositions, the package may containprinted information. The printed information can be provided on a label,or on a paper insert, or printed on the packaging material itself. Theprinted information can include information that identifies the activeagents in the package, the amounts and types of inactive ingredients, anindication of what condition(s) the pharmaceutical composition(s) isintended to treat, and instructions for taking the pharmaceuticalcomposition, such as the number of doses to take over a given period oftime, the order to take the compositions, and the like. Thus, in variousembodiments the disclosure includes a pharmaceutical composition of theinvention packaged in a packaging material and identified in print, onor in the packaging material, that the composition is for use in thetreatment or prophylaxis of any disease, condition or disorder that isrelated to a deterioration of neurons, an insufficiency of neurons, or adefect in the function of neurons. In another embodiment, instead of apharmaceutical composition, the disclosure includes a nutraceuticalformulation(s), and the printed material provides information about useof such a formulation(s) for improving cognitive function, memory, motorfunction, overall well-being, or the like.

The following specific examples are provided to illustrate theinvention, but are not intended to be limiting in any way.

EXAMPLE 1

This Example demonstrates that groups of the FDA-approved drugsdiscussed herein can convert human glial cells into neurons in vitro.

The results were obtained by treatment of human astrocytes (HA1800,ScienCell Inc.) with a combination of Certinib (50 nM, Cer), Crizotinib(125 nM, Cri), Lithium Chloride (2 mM, Li), Flurbiprofen (500 nM, Flu),Pirfenidone (PFD, 5 nM), and Vitamin C (50 ug/mL, VC) together, or indifferent combinations as indicated for six days, which converted humanastrocytes into neurons. Medium with drugs were changed every two days.14 days after drug addition, cells were immunostained for neuronalmarker NeuN. The micrograph shows many human glial cells converted intoneurons. FIG. 1A, Combination of Crizotinib, Certinib, Flurbiprofen,Lithium Chloride, and Vitamin C converted human glial cells intoneurons. FIG. 1B, Combination of Crizotinib, Flurbiprofen, LithiumChloride, Pirfenidone, and Vitamin C converted human glial cells intoneurons. FIG. 1C, combination of Crizotinib, Flurbiprofen, LithiumChloride, and Pirfenidone converted human glial cells into neurons. FIG.1D, combination of Crizotinib, Certinib, Flurbiprofen, and LithiumChloride, converted human glial cells into neurons. FIG. 1E, vehiclecontrol 0.2% DMSO, the solvent for Cer, PFD, Cri and Flu, showed veryfew NeuN+ cells after 14 days. FIG. 1F, Quantitative analyses showingsignificant increase of NeuN+ cell numbers after drug-treatment: CCFLV(56.0±12.9 NeuN+ cells per field, 20× lens, 0.1 mm2), CFLPV (47.8±4.7,NeuN+ cells per field), CFLP (33.3±4.3 NeuN+ cells per field), CCFL(32.4±1.3 NeuN+ cells per field), compared to DMSO control group(6.6±2.7 NeuN+ cells per field). ***P<0.001, *P<0.05, One-Way ANOVAfollowed with Dunnett's multiple comparisons test.

FIGS. 2A-FIG. 2B provide results showing that neurons converted by theindicated compounds can survive at least 2 months in culture. Inparticular, human neurons converted from astrocytes after CCFLV (FIG.2A) or CFLPV (FIG. 2B) treatment survived at least 2 months in culture,and showed mature neuronal markers MAP2 (light blue) and NeuN (Red).DAPI (dark blue) labels cell nucleus.

EXAMPLE 2

This Example provides a non-limiting example of an in vivoimplementation of an aspect of this disclosure using intracranialadministration of groups of compounds in adult mice, as well as in atransgenic mouse model of Alzheimer's disease. In particular, FIG. 3Ashows that an intracranial injection of 2 μl small-molecule cocktailincluding Crizotinib 50 μM, Flurbiprofen 0.2 mM, Pirfenidone 2 μM,Vitamin C 10 mg/ml and LiCl 0.4 M into the hippocampus of 3-month-old WTmice, promoted adult neurogenesis revealed by immunostaining of newbornneuron marker doublecortin (DCX, green) and cell proliferation markerKi67 (red). FIG. 3B. Intracranial injection of small molecule cocktailinto the hippocampus of 1-year-old adult WT mice, significantly promotedadult neurogenesis in dentate gyrus (DG), supported by the increasednumber of DCX-positive new neurons and Ki67-positive proliferatingcells. FIG. 3C. Intracranial injection of small molecule cocktail intothe hippocampus of 5-month-old transgenic mouse model of Alzheimer'sdisease, significantly increased the number of DCX-positive new neuronsin DG. FIG. 3D. In GFAP::GFP mice, astrocytes are labeled by GFP.Lineage-tracing assay indicate the DCX+ new neurons were induced fromGFP-labeled astrocytes by small-molecule cocktail. FIG. 3E, FIG. 3F, andFIG. 3G present quantitative analysis showing the number of DCX+ newneurons was increased by intracranial injection of small-moleculecocktail into the hippocampus of 3-month-old WT mice (FIG. 3E),1-year-old adult WT mice (FIG. 3F), and 5-month-old transgenic micemodel of Alzheimer's disease (FIG. 3G). Student t test, *P<0.05,**P<0.01, n=3 mice per group.

EXAMPLE 3

This Example provides a demonstration that intraperitoneal injection ofa group of the FDA-approved drugs described herein can also increaseadult neurogenesis in mouse brain. In particular, FIG. 4A and FIG. 4Bprovide representative images showing adult neurogenesis in thehippocampal dentate gyrus after intraperitoneal injection of vehiclecontrol (FIG. 4A, 20% Captisol), or compound group cocktail (FIG. 4B)including Crizotinib 50 μM, Flurbiprofen 0.2 mM, Pirfenidone 2 μM,Vitamin C 10 mg/ml and LiCl 0.4 M (3-month-old WT mice with the dose of0.1 ml/10 g weight, injected daily for 1 month). Seven days afterchemical treatment, mice were sacrificed and examined withimmunostaining of newborn neuron marker DCX. (FIG. 4C) Quantitativeanalysis revealed an increased number of DCX+ neurons treated by theFDA-approved drug cocktail. Student's t test, *P<0.05, n=2 pairs.

While the invention has been described through specific embodiments,routine modifications will be apparent to those skilled in the art andsuch modifications are intended to be within the scope of the presentinvention.

What is claimed is:
 1. A method for generating neurons comprisingcontacting glial cells with a group of compounds comprising acombination of at least three of Crizotinib (Cri), Flurbiprofen (Flu),Lithium Chloride (Li), Vitamin C (VC), Ceritinib (Cer) and Pirfenidone(PFD).
 2. The method of claim 1, wherein the group comprises four of thecompounds.
 3. The method of claim 1, wherein the group comprises five ofthe compounds.
 4. The method of claim 1 wherein the group consists offour of the compounds.
 5. The method of claim 1, wherein the groupconsists of five of the compounds.
 6. The method of claim 1, wherein thegroup comprises: i) Cer/Cri/Li/Flu/VC, ii) PFD/Cri/Li/Flu/VC, iii)Cer/Cri/Li/Flu, or iv) PFD/Cri/Li/Flu.
 7. The method of claim 1, whereinthe group consists of: i) Cer/Cri/Li/Flu/VC, ii) PFD/Cri/Li/Flu/VC, iii)Cer/Cri/Li/Flu, or iv) PFD/Cri/Li/Flu.
 8. The method of claim 7 whereinthe glial cells are in brain, or spinal cord, or peripheral nervoussystem, of a mammal.
 9. The method of claim 8, wherein the groupcomprises Cri/Flu/PFD/VC/Li.
 10. The method of claim 9 wherein the groupconsists of the Cri/Flu/PFD/VC/Li.
 11. The method of claim 8, whereinthe group is administered to an individual in need of the neurons due toa condition that comprises neuronal loss and/or glial scarring and/orneural injury, and or aging, or neurodegeneration, or microcephaly, orsevere seizure.
 12. The method of claim 8, wherein the individual is inneed of the neurons due to ischemic brain damage.
 13. The method ofclaim 8, wherein the individual has been diagnosed with or is suspectedof having Alzheimer's disease.
 14. A pharmaceutical compositioncomprising a group of compounds comprising Crizotinib (Cri),Flurbiprofen, Lithium Chloride (Li), and at least one additionalcompound selected from Vitamin C (VC), Ceritinib (Cer) and Pirfenidone.15. The pharmaceutical composition of claim 14, wherein the groupcomprises: i) Cer/Cri/Li/Flu/VC, ii) PFD/Cri/Li/Flu/VC, iii)Cer/Cri/Li/Flu, or iv) PFD/Cri/Li/Flu.
 16. The pharmaceuticalcomposition of claim 14, wherein the group comprises Cri/Flu/PFD/VC/Li.17. The pharmaceutical composition of claim 15, wherein the groupconsists of the Cri/Flu/PFD/VC/Li.
 18. An article of manufacturecomprising a pharmaceutical composition comprising a group of compoundscomprising at least three of Crizotinib (Cri), Flurbiprofen (Flu),Lithium Chloride (Li), Vitamin C (VC), Ceritinib (Cer) and Pirfenidone(PFD), the article of manufacture further comprising printed materialproviding an indication that the group of compounds is for use intreating a condition associated with a need for functional neurons. 19.The article of manufacture of claim 18, wherein group comprises i)Cer/Cri/Li/Flu/VC, ii) PFD/Cri/Li/Flu/VC, iii) Cer/Cri/Li/Flu, or iv)PFD/Cri/Li/Flu.
 20. The article of manufacture of claim 18, wherein thegroup comprises Cri/Flu/PFD/VC/Li.